Heat shock proteins (HSPs) of the hsp70, hsp90 and calreticulin families are abundant soluble intracellular proteins that elicit powerful immunological responses. These responses include components of both the innate and adaptive aspects of T cell responses as well as its down regulation. The ability of HSPs to chaperone peptides and engage internalizing receptors on antigen presenting cells are two key properties that allow them to elicit immune responses. A key role for HSPs in cross- priming of antigens during T cell responses was also envisaged and demonstrated in 2005. In this application we aim to (i) investigate the role of the HSP receptor, CD91, in cross-priming in the murine system, (ii) examine the mechanism of immunogenicity of alpha2-macroglobulin (a2M), a CD91 ligand, complexed either endogenously and artificially to various peptides, and (iii) test the role of serum levels of a2M on the ability of mice to mount immune responses. Aim 1: To investigate the role of the HSP receptor CD91 in cross-priming of T cell responses. a. Determine the ability of RAP (a competitive inhibitor of CD91) -expressing cells to cross present antigens and mount effective T cell responses;b. Creation of a CD91 conditional knock-out mouse and the characterization of immunological responses in this mouse. Aim 2: To investigate the mechanism of a2M-mediated immunogenicity and its application to immunotherapy of cancer and infectious disease. a. Examine the mechanism of co-stimulatory activation by a2M;b. Examination of the intracellular trafficking and processing of a2M -peptide complexes. c. Test the complexes of tumor derived-peptides with a2M in prophylaxis and therapy of cancers;d. Examination of endogenously formed a2M -peptide complexes as immunogens. Aim 3: To investigate the role of blood a2M levels on immune system. a. Test the effects of blood a2M levels on the ability of mice to elicit T cell responses after tumor inoculation;b. Test the effects of blood a2M levels on the ability of mice to elicit T cell responses after immunization with HSP-peptide complexes. PUBLIC HEALTH RELEVANCE: We have identified a role for heat shock proteins in cross-priming, an event that is central to the initiation of adaptive immune responses against cancer and infectious disease. In this proposal we aim to examine the HSP receptor(s) involved and how we may exploit this receptor by channeling other ligands, such as a2-macroglobulin through this pathway. This will allow one to generate novel therapeutic agents against cancer and infectious disease by targeting cell surface receptors.